Comparison of laboratory- and clinical-grade reagents in isolation and cultivation of human amniotic epithelial cells
Chika Takano1,2, Isamu Taiko3, Remi Kuwabara2,3, Ibrahim Fathi3, Toshio Miki3.
1Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan; 2Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan; 3Department of Physiology, Nihon University School of Medicine, Tokyo, Japan
Introduction: The human amniotic epithelial cell (hAEC) is a type of placental stem cell, which differentiates into all three germ layers. The hAECs have attracted attention as a new source of cell transplantation therapies and are considered to be suitable for clinical use from the following points. First, the hAECs are isolated from the delivered placenta and thus are readily available and easily procured without invasive procedures. Second, a sufficient number of hAECs can be obtained from a single human placenta. Third, hAECs do not possess tumorigenicity, unlike other pluripotent stem cells. Several preclinical studies using rodent models have been shown the therapeutic potential of hAECs in the past decade. Now in the effort of clinical translation, preparation of the clinical protocol requires optimization of utilizing the clinical-grade reagents. In this study, we conducted a comparison experiment between laboratory-grade and clinical-grade reagents in isolation and cultivation of hAECs.
Methods: hAECs were isolated from the placentae of 3 patients who underwent scheduled Caesarean sections. Laboratory- and clinical-grade reagents were compared in cell isolation and cultivation for each case. Regarding hAECs isolation, total cell number per membrane amount (cell yield) and cell viability was evaluated. With several surface markers including CD49f and CD326 specific to hAECs, CD105 for stromal cells, CD45 and GlyA for hematopoietic cells, and CD31 for endothelial cells, purity of the hAEC was analyzed using flow cytometry. The rest of the isolated cells were cryopreserved in liquid nitrogen for three months. Thawing cells were cultured with laboratory- and clinical-grade media to evaluate cell viability and plating efficiency. Cell proliferation was examined using Cell Counting Kit-8 at the time points of 1, 3, and 5 days.
Results: The yield and viability of the isolated hAECs were equivalents between the laboratory- and the clinical-grade reagents. The purity of the cells was also sufficient (over 95% in both treatments), as no contamination of stromal cells or endothelial cells was detected. The cultured cells were well attached to plastic dishes, resulting in comparable viability and cell proliferation between the two groups.
Conclusion: The clinical-grade reagents were as suitable for hAECs isolation and cultivation as the laboratory-grade reagents. Using this protocol, we next prepared the clinical-grade hAEC products in our cell processing facility, which should be an important step for novel cell transplantation therapies.
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